Isolation and characterization of human spermatogonial stem cells

Abstract

BackgroundTo isolate and characterization of human spermatogonial stem cells from stem spermatogonium.MethodsThe disassociation of spermatogonial stem cells (SSCs) were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4)-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established.ResultsThe cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells.ConclusionsThe two-step enzyme digestion (by type I collagenase and trypsin) process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

“…On the other hand, the enzymatic digestion protocols are relatively complex and timeconsuming. Further, the use of more numbers of enzymes might cause cell injury while less numbers increase the possibility of cell contamination [13,42]. Previously, in farm animals, attempts were made to isolate the SSCs using either four [10,11], three [14] or two [43] enzymes with variable success.…”

“…The SSCs have been isolated using two enzymes in human [12,13], three enzymes in buffalo [14] and four enzymes in cattle [11] as well as goat [10]. Use of more enzymes ensures higher purity but it takes more time and may affect the viability of the cells.…”

“…The differential plating is cost effective, but requires more time and compromises with the purity and concentration [20,21]. The density gradient protocol is considered to be a time saving and cost-effective method for the enrichment of isolated SSCs [13].…”

“…Among the livestock species, goats are often considered as unique for transgenesis because of their small body size, shorter gestation period, prolificacy and relatively higher protein content in their milk [29]. There are several reports of SSC culture in different species including mice [22,30], rat [31,32], cattle [11,33], buffalo [34,35] and human [13,36]. But the isolation and enrichment as well as establishment of long term culture system for SSCs have not yet been exploited substantially in goats except a very recent report [10].…”

“…Use of more enzymes ensures higher purity but it takes more time and may affect the viability of the cells. However, testicular disaggregation using less number of enzymes requires additional step of enrichment of isolated SSCs with high purity [13].…”